Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The patterns of DNA fragmentation were evaluated following a brief exposure (2 h) of the human ileocecal adenocarcinoma cell line, HCT-8, to several specific thymidylate synthase inhibitors, a quinazoline (ZD1694) and benz[cd]indole-containing molecule (AG-331). The magnitude and size of DNA fragmentation induced by the two agents were assessed by alkaline elution for DNA single-strand breaks (ssbs), and by pulsed- and constant-field gel electrophoresis for DNA double-strand breaks (dsbs). Both agents induced dose-dependent DNA dsbs. While AG-331 induced ssbs and dsbs only in nascent DNA, ZD1694 affected both genomic and nascent DNA. The fragments of newly synthesized and genomic DNA, estimated by pulsed-field gel electrophoresis assay, were associated with the bands in the range of 0.05 to 1.1 and 1.1 to 5.7 megabases, respectively. 5-fluoro-2'-deoxyuridine (FdUrd), like ZD1694, produced both mature and nascent DNA fragmentation, whereas only nascent DNA breakage induced by 5-fluorouracil (FUra) was detected, similar to AG-331. The induction of both mature and nascent DNA fragmentation by ZD1694 and FdUrd appears to correlate with the higher, but similar, potency of these agents. Aphidicolin, a DNA polymerase inhibitor, protects from DNA dsbs and cytotoxicity by ZD1694 and AG-331. These observations suggest that replicative DNA synthesis is an important factor in ZD1694- and AG-331-induced DNA fragmentation and, subsequently, cell growth arrest. The results indicate that although the new antimetabolites investigated herein were developed and extensively evaluated as specific and potent thymidylate synthase inhibitors, DNA damage appears to be an important additional determinant of drug effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contrasting patterns of DNA fragmentation induced by thymidylate synthase inhibitors, ZD1694 and AG-331. 757 30

We have reported that noncytotoxic concentrations of 3'-azido-3'-deoxythymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5'-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N6-[4-(Morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 microM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 microM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 microM AG-331 plus 1.8 microM [3H]-AZT produced as much as a 68% +/- 7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells.
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PMID:Cytotoxic and biochemical implications of combining AZT and AG-331. 785 Sep 19

Using four complementary approaches, ie., cell synchronization, bromodeoxyuridine labeling, and DNA and Western blot analyses, we investigated the underlying mechanism of cell cycle perturbation in response to ZD1694, a quinazoline-based antifolate thymidylate synthase inhibitor. With a single exposure at a concentration of 1 microM for 2 h, ZD1694 completely inhibits thymidylate synthase over 72 h and causes a sustained growth for at least 120 h, DNA damage, and p53 induction in human carcinoma cells. Although these cells displayed an S-phase block with the precise terminal arrest point depending on the timing of drug treatment in the cell cycle, their DNA-replicating machinery associated with polymerase alpha was preserved intact. When supplemented with exogenous dThd, these cells resumed an apparently normal S-phase progression for at least 4 h. Kinetic analyses based on synchronized cells indicate that S-phase arrest occurs first, preceding the induction of DNA double strand breaks and p53/p21. SW480 cells, in which p53mu failed to transduce p21, also exhibited the mode of S-phase arrest, essentially indistinguishable from that displayed by HCT-8 cells expressing the functional p53 (p53wt). That the DNA replication process is prerequisite for DNA double strand breaks was indicated by the following: (a) DNA damage occurred only when cells treated with ZD1694 progressed through S phase; and (b) the inhibition of DNA polymerase alpha by aphidicolin-blocked DNA damage. Based on the above, we conclude that S-phase arrest by ZD1694, with a subsequent damage of DNA double strands, is caused by the block of DNA synthesis in the middle of replication due to dTTP depletion and not by p53-mediated G1-G2 checkpoint mechanisms or p21-induced inactivation of the DNA-replicating machinery.
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PMID:DNA damage and p53 induction do not cause ZD1694-induced cell cycle arrest in human colon carcinoma cells. 884 Sep 89

New inhibitors of the enzyme thymidylate synthase (TS) are now reaching clinical application. Alteration of the dUTP: dTTP ratio may be critical to TS inhibition-induced tumor cell death. The DNA polymerase assay with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts of HT29 human colon carcinoma cells treated with the specific TS inhibitor ZD1694 [N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid]. These results revealed an increase in the dUTP:dTTP ratio at 2 hr after a 2-hr exposure to ZD1694 at concentrations of 0.05 to 0.2 microM with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhibition. This assay is highly sensitive and reproducible for levels of dUTP and is less labor intensive than traditional assays.
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PMID:Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate synthase-inhibited cells using a modified DNA polymerase assay. 933 81

Thymidylate synthase (TS) is an important target of several classes of chemotherapeutic agents. Although the precise mechanism of cytotoxicity in thymidylate deprivation remains obscure, uracil misincorporation and DNA strand breaks are recognized as important events during thymidylate deprivation. Base excision repair (BER) plays a primary role in removing damaged or modified bases from the genome, including uracil. Because of uracil misincorporation, BER is hypothesized to play a role in the cellular response to thymidylate deprivation. In this study, we used murine embryo fibroblasts wild-type or homozygous null for DNA polymerase beta (beta-pol), which plays a central role in BER. We found that, compared with wild-type, beta-pol null cells were resistant to the toxic effects of raltitrexed (Tomudex, ZD1694), a folate inhibitor of TS. There was little difference in TS levels or in TS-ligand complex formation between the cell lines. Furthermore, cells deficient in XRCC1, a scaffold protein for the final steps of BER, were also modestly resistant to raltitrexed compared with XRCC1-proficient cells. Cell cycle analysis revealed that the responses of the wild-type and beta-pol null cells were similar during drug exposure. However, following drug removal, the beta-pol null cells appeared to resume cell cycle progression more rapidly than the wild-type cells. The results suggest that BER plays a role in modulating the toxic effects of TS inhibitors, and that this role occurs during recovery from TS inhibition.
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PMID:Involvement of base excision repair in response to therapy targeted at thymidylate synthase. 1521 Aug 61